Ultrasonographic findings in cows with left displacement of abomasum, before and after reposition surgery.

BACKGROUND: The natural incidence of left displacement of abomasum (LDA) in dairy cows was high. The diagnosis of LDA usually relies on characteristic physical exam findings but that transabdominal ultrasound is a useful technique that has been applied to the diagnosis of gastrointestinal diseases of dairy cows in equivocal cases. METHODS: Forty dairy cows with LDA were clinically and ultrasonographically examined to determine the position and the echogenic property of the abomasum. The cows were examined ultrasonographically on the left side, from the 9th intercostal space (ICS) to the 12th ICS as well as the ventral left abdomen before and after reposition surgery. RESULTS: The vital signs were within normal range in most of the cows and the 'pinging' were clearly heard in 37 cows. The abomasal gas cap was visualized from the 9th to 12th ICS in 37 cows and characterized by reverberation artifacts. The abomasal ingesta appeared as homogeneous hypoechoic fluid with scattered hyperechoic foci and were mainly visible in the median region and ventral region of the 9th to 11th ICS in 35 cows. The pyloric canal was detected from the ventral left abdomen wall in 30 cows and appeared as a loop with hypoechogenic wall and echogenic luminal contents in cross section. CONCLUSION: These typical ultrasonograms, including reverberation artifacts, homogenous hypoechoic structures, are important diagnostic feature in ultrasonography of LDA. Furthermore, the circular acoustic image structure of the pyloric canal is an important characteristic of LDA, so it can be used as an important diagnostic basis of LDA.

Berberine Protects against NEFA-Induced Impairment of Mitochondrial Respiratory Chain Function and Insulin Signaling in Bovine Hepatocytes.

Fatty liver is a major lipid metabolic disease in perinatal dairy cows and is characterized by high blood levels of non-esterified fatty acid (NEFA) and insulin resistance. Berberine (BBR) has been reported to improve insulin sensitivity in mice with hepatic steatosis. Mitochondrial dysfunction is considered a causal factor that induces insulin resistance. This study investigates the underlying mechanism and the beneficial effects of BBR on mitochondrial and insulin signaling in bovine hepatocytes. Revised quantitative insulin sensitivity check index (RQUICKI) of cows with fatty liver was significantly lower than that of healthy cows. Importantly, the Akt and GSK3ß phosphorylation levels, protein levels of PGC-1α and four of the five representative subunits of oxidative phosphorylation (OXPHOS) were significantly decreased in cows with fatty liver using Western Blot analysis. In bovine hepatocytes, 1.2 mmol/L NEFA reduced insulin signaling and mitochondrial respiratory chain function, and 10 and 20 umol/L BBR restored these changes. Furthermore, activation of PGC-1α played the same beneficial effects of BBR on hepatocytes treated with NEFA. BBR treatment improves NEFA-impaired mitochondrial respiratory chain function and insulin signaling by increasing PGC-1α expression in hepatocytes, which provides a potential new strategy for the prevention and treatment of fatty liver in dairy cows.

BHBA suppresses LPS-induced inflammation in BV-2 cells by inhibiting NF-κB activation.

ß-Hydroxybutyric acid (BHBA) has neuroprotective effects, but the underlying molecular mechanisms are unclear. Microglial activation plays an important role in neurodegenerative diseases by producing several proinflammatory enzymes and proinflammatory cytokines. The current study investigates the potential mechanisms whereby BHBA affects the expression of potentially proinflammatory proteins by cultured murine microglial BV-2 cells stimulated with lipopolysaccharide (LPS). The results showed that BHBA significantly reduced LPS-induced protein and mRNA expression levels of iNOS, COX-2, TNF-α, IL-1ß, and IL-6. Blocking of GPR109A by PTX resulted in a loss of this anti-inflammatory effect in BV-2 cells. Western blot analysis showed that BHBA reduced LPS-induced degradation of IκB-α and translocation of NF-κB, while no effect was observed on MAPKs phosphorylation. All results imply that BHBA significantly reduces levels of proinflammatory enzymes and proinflammatory cytokines by inhibition of the NF-κB signaling pathway but not MAPKs pathways, and GPR109A is essential to this function. Overall, these data suggest that BHBA has a potential as neuroprotective drug candidate in neurodegenerative diseases.

Dietary pseudopurpurin effects on bone mineral density and bone geometry architecture in rats.

The objective of our study was to evaluate whether feeding pseudopurpurin affects bone mineral density and bone geometry architecture in rats. Pseudopurpurin was extracted, analyzed and purified using an HLPC-ESI-MS. Rats were given 0% and 0.5% pseudopurpurin powder in their diet. Femurs of rats were examined at 0.5, 1 and 2 months after pseudopurpurin feeding. Compared with rats in the group 0%, the bone mineral density, and the calcium, magnesium, zinc and manganese concentrations in the rats femur in the group 0.5% increased significantly at 1 month and 2 months after pseudopurpurin feeding. Analytical results of micro-computed tomography showed that the group 0.5% displayed an increase in the trabecular volume fraction, trabecular thickness and trabecular number of the distal femur at 1 and 2 months after pseudopurpurin feeding, and the mean thickness, inner perimeter, outer perimeter, and area of the femur diaphysis were significantly increased at 2 months after pseudopurpurin feeding compared with the group 0%. In parallel, the trabecular separation and structure model index of the distal femur were decreased, compared with the group 0% at 1 and 2 months after pseudopurpurin feeding. In the 0.5% and 0% groups, there was no damage to kidney and liver by histopathology analysis. The long-term feeding of pseudopurpurin is safe for rats. The feeding of 0.5% pseudopurpurin which has specific chemical affinities for calcium for bone improvement and level of bone mineral density, enhances the geometry architecture compared with the 0% group.

Determining prognostic factors and optimal surgical intervention for early-onset triple-negative breast cancer.

Background: Few studies have focused specifically on prognostic factors and optimal surgical intervention for early-onset triple-negative breast cancer (eTNBC), which is characterized by high malignancy and poor prognosis. Methods: We performed a cohort study with a median follow-up of 31 months using Surveillance, Epidemiology, and End Results (SEER) data of patients diagnosed with stages I-III eTNBC between 2010 and 2016. In addition, we collected cases between 2006 and 2016 from our center as an external validation set. Clinical features, pathologic characteristics and oncologic outcomes were analyzed. Prognostic factors for overall survival (OS) and breast cancer-specific survival (BCSS) were determined by Cox proportional hazards analyses and were incorporated into the prognostic nomogram. Subgroup analysis based on propensity score matching method was conducted to explore the subset of patients that would benefit from breast-conserving therapy (BCT). Results: Based on SEER dataset, patients with eTNBC were more likely to undergo mastectomy than BCT. On multivariable analysis, patients with better survival outcomes were those not married, uninsured, had higher T and N stage, and had histological type of mixed invasive ductal and lobular carcinoma. The prognostic nomogram based on these variables successfully predicted the 3- and 5-year BCSS (C-index in training cohort, 0.774; in validation cohort from SEER, 0.768; in validation cohort from our center, 0.723). Subgroup analysis illustrated that patients with T1N0M0 or T2-4N+M0 tumors who underwent BCT achieved longer overall survival than those who underwent mastectomy (for T1N0M0, P = 0.022; for T2-4N+M0, P = 0.003); however, the type of surgery did not influence OS among patients with T1N+M0 or T2-4N0M0 tumors (for T1N+M0, P = 0.305; for T2-4N0M0, P = 0.317). Conclusions: The prognosis of patients with eTNBC is mainly affected by marital status, insurance status, T stage, N stage and histological type. The prognostic nomogram based on these factors is quite reliable. Subgroup analysis suggested that BCT may be a superior option for patients with eTNBC, especially those with T1N0M0 and T2-4N+M0 tumors.

Prognostic Factors and a Nomogram Predicting Survival in Patients with Breast Ductal Carcinoma in situ with Microinvasion: A Population-Based Study.

PURPOSE: Ductal carcinoma in situ with microinvasion (DCISM) can be challenging to balance the risks of overtreatment versus undertreatment. We aim to identify prognostic factors in patients with DCISM and construct a nomogram to predict breast cancer-specific survival (BCSS). MATERIALS AND METHODS: A retrospective cohort study of women diagnosed with DCISM from 1988 to 2015 who were identified in the Surveillance, Epidemiology and End Results database. Clinical variables and tumor characteristics were evaluated, and Cox proportional-hazards regression was performed. A nomogram was constructed from the multivariate logistic regression to combine all the prognostic factors to predict the prognosis of DCISM patients at 5 years, 10 years, and 15 years. RESULTS: We identified 5438 total eligible breast cancer patients with a median and max survival time of 78 and 227 months, respectively. Here, patients with poorer survival outcomes were those diagnosed between 1988 and 2001, African-American race, under 40 years of age, higher tumor N stage, progesterone receptor-negative tumor, and received no surgery. The nomogram was constructed by the seven variables and passed the calibration and validation steps. The area under the receiver operating characteristic (ROC) curve (AUC) of both the training set and the validating set (5-year AUC: 0.77 and 0.88, 10-year AUC: 0.75 and 0.73, 15-year AUC: 0.72 and 0.65). Receiving chemotherapy was associated with a better BCSS (hazard ratio, HR=0.45, 95% confidence interval, 95% CI = 0.23-0.89), especially in patients with estrogen receptor (ER) negative, progesterone receptor (PR) negative (HR = 0.35, 95% CI = 0.13-0.97) and ER+PR-/ER-PR+ DCISM (HR = 0.07, 95% CI = 0.01-0.59). CONCLUSION: Our current study is the first to construct nomograms of patients with DCISM which could help physicians identify breast cancer patients that more likely to benefit from more intensive treatment and follow-up. Chemotherapy might benefit patients with ER-PR- and ER+PR-/ER-PR+ DCISM.

Nuciferine improves high-fat diet-induced obesity via reducing intestinal permeability by increasing autophagy and remodeling the gut microbiota.

Nuciferine (NF) has received extensive attention due to its medicinal value in the treatment of metabolic diseases, such as obesity; however, to date, the effects of NF on obesity-related intestinal permeability, autophagy and the gut microbiota have not been investigated. Herein, C57BL/6J mice were fed either a chow or a high-fat diet (HFD) with or without NF for 8 weeks. The results showed that NF supplement reduced weight gain, fat accumulation and intestinal permeability in the HFD mice accompanied by improved autophagy. Subsequently, an in vitro experiment was performed using Caco-2 and HT-29 cells, which showed that NF supplement not only promoted the formation of autophagosomes and autophagolysosomes, but also alleviated LPS-increased intestinal permeability. Importantly, NF supplement protected from LPS-induced paracellular permeability impairment after the administration of autophagy-related gene (Atg) 5 small-interfering RNA (siRNA). These results demonstrate that NF exerts beneficial effects on the intestinal permeability by improving autophagy. Furthermore, we also found that NF supplement lowered the abundance of Butyricimonas and increased the abundance of Akkermansia, an anti-obesity bacterium. Thus, overall, we demonstrated that NF supplement confers reduced intestinal permeability by improving autophagy and alters the composition of the gut microbiota in HFD-fed mice, thereby producing an anti-obesity effect.

Diagnostic Value of Transthoracic Echocardiography in Patients With Anomalous Origin of the Left Coronary Artery From the Pulmonary Artery.

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital coronary abnormality associated with early infant mortality and sudden death in adults. Transthoracic echocardiography (TTE) plays an important role in early detection and diagnosis of ALCAPA as a noninvasive modality. However, its diagnostic value is not well studied. The purpose of this study is to determine the performance of TTE in the diagnostic assessment of ALCAPA as compared with coronary CT and invasive coronary angiography. A total of 22 patients (13 women and 9 men, mean age, 12.9 ±â€Š19.5 years) with ALCAPA who underwent echocardiographic examination for clinical diagnosis were retrospectively reviewed and analyzed. Transthoracic echocardiographic features of ALCAPA were analyzed and its diagnostic value was compared with invasive coronary angiography and coronary CT angiography (CTA) with surgical findings serving as the gold standard. Surgery was performed in all of the patients to establish the dual coronary artery system. Five underwent the Takeuchi procedure and 17 had re-implantation of the anomalous left coronary artery. Of 20 patients, echocardiographic diagnoses were in good agreement with findings at surgery, resulting in the diagnostic accuracy of 90.9%. Two cases were misdiagnosed-one as the right coronary artery to pulmonary artery fistula and the other as rheumatic heart disease. The echocardiographic features of these patients with ALCAPA included: abnormal left coronary ostium arising from the pulmonary trunk with retrograde coronary artery flow in 20 patients; enlargement of the right coronary artery in 17 patients; abundant intercoronary septal collaterals in 17 patients; and moderate and significant mitral regurgitation in 14 patients. The diagnostic accuracy of invasive coronary angiography (in 17 patients) and coronary CTA (in 9 patients) was 100%. This study shows that TTE is an accurate, noninvasive imaging modality for displaying the origin of coronary arteries and demonstrating the coronary courses as well as other associated abnormalities in patients with ALCAPA.

Preparation of novel monoclonal antibodies against chelated cadmium ions.

The detection of cadmium ions using enzyme-linked immunosorbent assays (ELISA) has been reported by several research groups. Because cadmium ions are too small to stimulate the immune system, high molecular weight immunogens of cadmium are constructed using bifunctional chelators. At present, the most commonly used bifunctional chelator for the preparation of antigens for heavy metal ions is 1-(4-isothiocyanobenzyl) ethylenediamine N,N,N',N'-tetraacetic acid (ITCBE). However, the price of ITCBE is high. So we are interested in a cheaper bifunctional chelator, 1-(4-aminobenzyl) ethylenediamine N,N,N',N'-tetraacetic acid (aminobenzyl-EDTA). Here, cadmium ions were conjugated to carrier proteins using aminobenzyl-EDTA to make artificial antigens. Then, several mice were immunized with the antigen. And monoclonal antibodies (MAbs) against cadmium were produced. Spleen cells of immunized mice were fused with myeloma cells. The resulting hybridomas were screened using protein conjugates which were covalently bound to metal-free EDTA or cadmium. Three hybridoma cell lines (A3, E4 and B5) that produced MAbs with high selectivity and sensitivity were expanded for further study. Cross-reactivities with other metals were below 1 %. These antibodies were used to construct competitive ELISAs. The IC50 for A3 was 8.4 µg/l. The detection range and the lowest detection limit using the antibody A3 was 0.394-64.39 and 0.051 µg/l, respectively. Spike-recovery studies in tap water showed that the antibody A3 could be used for cadmium detection in drinking water.

An updated method for the isolation and culture of primary calf hepatocytes.

Primary hepatocytes are commonly used during in vitro studies, but care must be taken with isolation and culture of the cells to ensure their viability. In this study, hepatocytes were isolated from the liver (caudate process) of a newborn calf by the collagenase perfusion and digestion method. The trypan blue exclusion method was used to determine total cell number and the survival rate of hepatocytes, while hepatocyte function was assessed by measuring lactate dehydrogenase, albumin and urea in culture medium supernatants at 24, 48, 72, 96, 120, 144 and 168 h. Results showed that the number of viable cells/g of liver (wet weight) averaged 1.12×10(7) cells/g, with an average hepatocyte viability of 85.7% (range 83-92%). After 48 h of culture, the hepatocytes solidly adhered to the well culture plate and were spread in an epithelioid shape, with clear cell boundaries between the cells and biliary ductule-like structures formed which persisted for up to 10 days. Hepatocyte function was optimal at 72 h after isolation and culture. This simple and economical procedure for the isolation and culture of viable cells may be useful for in vitro bovine hepatocyte studies.

A colloidal gold probe-based silver enhancement immunochromatographic assay for the rapid detection of abrin-a.

High-affinity anti-abrin-a monoclonal and polyclonal antibodies were used to develop a sandwich immunochromatographic assay and silver enhancement technology was used to further increase the sensitivity. Using a matrix of double distilled water or soybean milk with added abrin-a, the visual detection limit was found to be 10 ng mL(-1). The detection limit was 0.1 ng mL(-1) for abrin-a, an increase in sensitivity of 100-fold when the silver enhancement technology was employed. The assay was portable and very simple to perform and the detection was completed within 20 min without complicated handling procedures. There was no significant cross-reactivity with several homologous toxins and associated agglutinin. The assay reagents could be stored for 12 weeks at 4°C without significant loss of activity. These characteristics make the strip assay to be an ideal candidate for the development of a rapid toxin detection kit.

Preparation and identification of monoclonal antibody against abrin-a.

BALB/c mice were immunized four times with formalin-prepared abrin-a. Using the polyethylene glycol method, immunized splenocytes were isolated and fused with SP2/0 cells. An indirect ELISA was established and used to detect positive clones secreting monoclonal antibodies (mAbs) against abrin-a. After analysis, three hybridoma clones secreting IgG-subtype mAbs were obtained. The antibodies were purified from the hybridoma growth medium using protein A or G affinity chromatography. Western blot analysis was used to analyze the antigenic epitopes on abrin-a recognized by the mAbs. The mAbs were specific for abrin-a, with no detectable cross-reactivity with several homologous toxins and associated agglutinins. Sandwich ELISA was then developed using these mAbs, which had a detection limit for abrin-a of 7.8 ng/mL.

[In situ hybridization study on the expression of Kiss-1 and KAI-1 metastasis suppressor genes in gastric cancer].

OBJECTIVE: To investigate the mRNA levels of Kiss-1 and KAI-1 metastasis suppressor genes in gastric cancer, and to explore its clinical value. METHODS: In situ hybridization was used on routinely paraffin-embedded sections of resected specimens of 49 cases with gastric cancer and 20 cases with pericancerous tissue. RESULTS: The positive rates and the scores of Kiss-1 and KAI-1 mRNA in gastric cancer tissue were significantly lower than those in pericancerous tissue (P<0.01). The positive rates and scores in normal to mild-atypical hyperplasia cases were significantly higher than those in middle to severe-atypical hyperplasia of pericancerous tissue (P<0.05,P<0.01). The positive rates and scores of Kiss-1 and KAI-1 mRNA in patients with infiltrating depth T1~T2, without lymph node metastasis, with only first group of lymph node metastasis, and without distant organ metastasis were significantly higher than those in patients with infiltrating depth T3~T4, with lymph node metastasis, with second or third group of lymph node metastasis and with distant organ metastasis. A strong positive correlation was found between the expressive scores of Kiss-1 and KAI-1 mRNA in gastric cancer (r=0.53, P<0.01). CONCLUSION: The expression of Kiss-1 and/or KAI-1 mRNA may be important biological markers of reflecting invasive and metastatic potential and prognosis in gastric cancer. The assays of Kiss-1 and/or KAI-1 mRNA expression level in benign lesions of stomach may have important clinical value for the protection and early-stage finding of gastric cancer.